Abstract

Breast cancer is the most common cancer among women worldwide. Breast cancer metastasis primarily happens through lymphatic system, where the extent of lymph node metastasis is the major factor influencing staging, prognosis and therapeutic decision of the disease. We aimed to study the protein expression changes in different N (regional lymph nodes) stages of breast cancer. Protein expression profiles of breast cancerous and adjacent normal tissues were mapped by proteomics approach that comprises of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry (LC-MS/MS) analysis. Calreticulin and tropomyosin alpha 3 chains were the common up-regulated proteins in N0, N1 and N2 stages of breast cancer. Potential biomarker for each N stage was HSP 70 for N0, 80 k protein H precursor and PDI for N1 stage while 78 kDa glucose-regulated protein was found useful for N2 stage. In addition, significant up-regulation of PDI A3 was detected only in the metastasized breast cancer. The up-regulation expression of these proteins in cancerous tissues can potentially use as indicators for diagnosis, treatment and prognosis of different N stages of breast cancer.

Keywords: Breast cancer, lymph nodes, proteomic, 2D-PAGE, LC-MS/MS.

Introduction

Breast cancer is a malignant disease originated from breast's cell, where it usually forms from the terminal duct-lobular unit that supplies milk 1. In 2008, 460,000 deaths due to breast cancer were reported worldwide 2.

Infiltrating ductal carcinoma (IDC) is the most common and aggressive type of breast cancer 3. It is originated in the lactiferous duct, where it can break through the duct tube and invade or infiltrate the surrounding breast tissue, it can also spread to other parts of the body through lymph and blood systems 4.

Metastasis is defined as the process by which tumor cells spread from primary tumor site to secondary site (nearby or distant organ) and proliferate. Metastasis can occur through blood or lymph node 5. In breast cancer, metastasis primarily happens through the lymphatic system 6. The extent of lymph node (N) metastasis is the major factor influencing staging and prognosis of most malignancies and often determines therapeutic decision 7. Therefore, we aimed to study protein expression changes of N stages of IDC breast tissues as classified by the TNM staging system, where emphasis will be given to the up-regulated proteins in cancerous tissues. Protein expression profiles for breast cancerous and its adjacent normal tissues were mapped by using proteomics approach. We hoped to identify biomarkers that can be used as diagnostic or therapeutic markers for different stages of breast cancer.

Materials and methods

Tissue collection: Human ethical approval was obtained from the Human Ethical Clearance Committee of Universiti Sains Malaysia (USM) and the Ministry of Health Malaysia. Patient consents were obtained from the patients or their next of kin before collection of tissue specimens. Thirty-eight pairs of breast tissues comprised of normal and cancerous tissues were collected from Penang General Hospital (GH) after being confirmed as normal and cancerous, respectively by hospital pathologist. The tumor type used was IDC. There were 13 patients in N0 stage, 13 patients in N1 stage and 12 patients in N2 stage according to the TNM staging system (Table ​(Table1).1). The patients did not receive any preoperative neo-adjuvant chemotherapy or radiotherapy prior to surgery.

Tissue processing: The tissue specimens were stored frozen at -80°C until analysis. Prior to protein extraction, tissues were thawed to room temperature and then rinsed thoroughly with distilled water. Fat tissues were removed from the tissue specimens and the specimens were cut into small pieces and transferred to microcentrifuge tubes.

Protein extraction: An amount of 250 mg of tissue was subjected to sequential extraction using Tris Buffer (TRIS) and Thiourea Lysis Buffer (TLB). First, a volume of TRIS (40 mM Tris, 1 mM protease inhibitor and 2 mM benzonase) was added to the tissue in a microcentrifuge tube following a ratio of 1:2; tissue (weight): buffer (volume). The tissue was then disrupted on ice for 10 minutes using a plastic homogenizer. The lysate was vortex for 30 seconds and centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatant was recovered and kept at -80°C for other analysis. Subsequently, 250 μL of TRIS was added to the remaining pellet and vortex briefly. The mixture was then centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatant was discarded. TLB [8 M Urea, 2 M Thiourea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.4% (w/v) carrier ampholyte, 50 mM DTT and 1 mM AEBSF] was added to the remaining pellet following a ratio of 1:1 pellet (weight): buffer (volume). The mixture was mixed thoroughly by brief vortexing and centrifuged at 13000 rpm for 15 minutes at 4°C. After centrifugation, the supernatant was collected in a microcentrifuge tube and subjected to protein concentration determination using RC-DC protein assay (Bio-Rad, USA),

Image analysis: All 2D images were captured using VersaDoc Imaging System (Bio-Rad, USA) and analyzed using PDQuest Software version 7.3 (Bio-Rad, USA). Match sets consisted of 2D-gel images of protein extracts from normal and cancerous tissues of the same patient were created. The common and differentially expressed protein spots between normal and cancerous tissues were compared and identified. Normalization was applied to all the captured gel images, using this device, the intensity of every protein spots were measured as proportion to the total density of valid spots. Wilcoxon paired-sample test was used to determine the statistical significance in the difference in intensity between protein spots from normal and cancerous tissues.

In-gel digestion: All targeted protein spots were subjected to in-gel digestion according to Gam et al. (2003). It is a sample preparation procedure prior to LC-MS/MS analysis. Spots containing proteins were excised from gel and rinsed thoroughly with deionized water to remove excess SDS. The gel pieces were washed with 100 mM ammonium bicarbonate (NH4HCO3) for 10 minutes. The buffer was discarded. Acetonitrile (ACN) was added onto the gel pieces for dehydration purpose and discarded after 5 minutes incubation. Then, the gel pieces were subjected to vacuum centrifugation (Eppendorf, Germany) until dried. These steps were repeated four times. Then, 15 μL of 10 ng/μL trypsin prepared in digestion buffer (50 mM NH4HCO3, 5 mM CaCl2) was added to the gel pieces and incubated on ice for 45 minutes. The excess digestion buffer containing trypsin was discarded and 10 μL of digestion buffer without trypsin was added and incubated at 37°C overnight. After centrifugation at 2000 rpm for 2 minutes at room temperature, the supernatant were collected and kept in microcentrifuge tube. Subsequently, 15 μL of 20 mM NH4HCO3 was added to the gel pieces and incubated at room temperature for 10 minutes. The supernatant was collected and pooled with the previous collection after a brief spin. Then, the remaining peptides were further extracted using four repeated extraction cycles using 5% (v/v) formic acid in 7:3 ACN: dH2O. In each cycle, gel pieces were incubated for 20 minutes in the buffer and spun down. All the supernatants recovered were pooled with the previous collections. The pooled peptide extracts were evaporated until dried using continuous flow of nitrogen gas.

Reverse phase high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS): The peptides were reconstituted in 30 µL of 0.1% (v/v) formic acid in 85:15 solution of dH2O: ACN. The mixture was briefly spun and the supernatant was recovered and transferred into a polypropylene vial insert and placed in autosampler of a HPLC system. A sample volume of 15 µL was injected into the enrichment column (Zorbax SB C18, 35 X 0.5 mm, particle size 5 µm) at an isocratic flow rate of 0.05 mL/min using a binary pump HPLC 1100 Series (Agilent, Germany). The mobile phase was 0.1% (v/v) formic acid in 97:3 dH2O: ACN. Then, the concentrated peptides were eluted and transferred to a reverse phase column (Zorbax 300SB C18, 150 X 0.3 mm, particle size 5 µm) by using a capillary pump operated at flow rate of 4 µL/min. The peptides were eluted at a linear gradient of 5% B to 95% B in 70 minutes and held constant at 95% B for 5 minutes. Mobile phase A was 0.1% (v/v) formic acid in deionized water while mobile phase B was 0.1% (v/v) formic acid in ACN. The HPLC was interfaced to an electrospray ionization (ESI)-ion trap mass analyzer. The MS data was acquired through data dependent analysis method consisted of full scan MS and MS/MS scan. The eluted peptides from HPLC were ionized by ESI operating in positive ion mode. The two most intense positively charged ions in every MS scan that exceeded the minimum threshold of 3000 counts were isolated and excited to MS/MS scan. In MS/MS scan, the peptide was subjected to collision induced dissociation (CID) to produce MS/MS spectrum. The MS parameters used were: dry gas flow rate of 6 L/min, dry gas temperature of 300ºC, nebulizer gas flow rate of 15 L/min and nebulizer pressure of 15.0 psi. The MS/MS analysis was performed with these parameters: collision energy at 1.15 V, capillary voltage of 3.5 kV, exit capillary voltage of 84.5 V, skimmer 1 voltage of 17.2 V, skimmer 2 voltages at 6.0 V, scan range of 200-1800 m/z with a scan time of 1 second, charge state of 2, and isolation width of 2 m/z.

Protein identification: The MS/MS data obtained was used to interrogate the identity of protein through Mascot Protein Database (MSDB) search engine. The parameters used were Homo sapiens for taxonomy, trypsin for enzyme, carboxymethyl for fixed modification. The peptide tolerance and MS/MS tolerance were +/- 2 Da and 0.8 Da, respectively. The instrument type was set as ESI-TRAP and only one missed cleavage was allowed. The function and characteristic of proteins were obtained from Swiss-Prot (www.expasy.org) and NCBI (www.ncbi.nlm.nih.gov) protein database. The protein name, score, molecular weight (MW), isoelectric point (pI), and sequence coverage (seq. cov) were obtained through MASCOT Protein Database (MSDB) search engine (www.matrixscience.com). The Swiss-Prot number was obtained through NCBI blast (www.ncbi.nlm.nih.gov). The grand average of hydropathy (GRAVY) was obtained through Expasy proteomic server (expasy.org). GRAVY is a value calculated by sum of the hydropathy value for each amino acid and dividing by number of residue in protein sequence. It ranged from -4 to +4, where the more positive value means the protein is more hydrophobic and vice versa for protein with negative value 8. The function of proteins was obtained through www.uniprot.org.

Western Blot: Western blotting was performed according to Towbin et al., (1979) with modifications. The tissue extract was separated using SDS-PAGE at constant voltage of 200 V. After SDS-PAGE, the gel was soaked in cold transfer buffer [25 mM Tris, 192 mM Glycine, 1.3 mM SDS and 20% (v/v) methanol] for 30 minutes while nitrocellulose membrane and blotting papers of 8.0 x 7.4 cm in size (Bio-Rad, USA) were soaked in transfer buffer for 1 hour. The pore size of nitrocellulose membrane was 0.45 µm. Semi-dry blotting method (Lauriere, 1993) was applied using TE Semiphor semi-dry transfer unit (Hoefer Scientific, Germany). The transfer of proteins from gel to nitrocellulose membrane was conducted at 134 mA for 1.5 hours. After this, the membrane was rinsed with washing buffer [0.1% (w/v) BSA, 0.1% (v/v) Tween 20 in PBS] and blocked with blocking buffer [3% (w/v) BSA, 0.1% (v/v) Tween 20 in PBS] with gentle agitation for 2 hours at room temperature. The blocking buffer was discarded and the membrane was washed three times with washing buffer. The membrane was then incubated with primary antibody overnight at 4ºC with gentle agitation. This is followed by incubation with secondary antibody for 2 hours at room temperature after being washed three times with washing buffer. The membrane was incubated with substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad, USA) with gentle shaking for 15 minutes in order to visualize the interaction between targeted protein and its antibody (primary antibody).

Result

In this study, a total of 38 pairs of normal and cancerous breast tissues were subjected to identical protein extraction and 2D-PAGE procedures. All 2D-images were analyzed using gel image analyzer. Table ​Table11 shows basic information of all patients.

Inconsistent expression of a few proteins between patients was observed although a general consistent pattern of proteomes can be identified within normal and cancerous tissues, respectively. Hence, only the protein spots that were consistently expressed in greater than 80% of all patients in the same cohort were considered as valid protein spot. Using this approach, we hoped to minimize the identification of invalid protein spots that may result from variation between gels or error in the image analysis. The consistently expressed protein is more likely to be involved in the process of transformation of normal cell into cancer cells. A protein spot was categorized as up-regulated when the intensity of the spot in cancerous tissue is at least 2 folds or greater than that of normal tissue. The vice-versa was applied to down-regulated proteins. A protein was termed as up-regulated protein when it showed consistent up-regulation characteristic in greater than 65% patients in the cohort. The similar was applied to the down-regulated proteins.

Sixty-four valid protein spots on 2D gel images of TLB extracts of breast tissues were detected. The protein spots that showed consistent differentially expressed in >65% patients were subjected to in-gel digestion, LC-MS/MS analysis and MASCOT search. Figure ​Figure11 shows the up- and down-regulated protein spots on 2D gel image of a TLB extract from breast cancerous tissue. An example of MS and MS/MS spectrum of calreticulin are shown in Figure ​Figure2.2. The identities and characteristics of the significant up- and down-regulated proteins are shown in Table ​Table22.

The protein identity as identified by LC-MS/MS and MASCOT database search engine was validated using Western blot experiments. All the potential biomarker with commercially available antibodies were subjected to this analysis, these biomarkers included calreticulin, PDI A3, HSP 60 and HSP 70, where the identify of all of these proteins were confirmed by Western blotting. Figure ​Figure33 shows the Western blot images for calreticulin and PDI A3 in breast normal and cancerous extracts from the same patient while beta-actin was used as an internal control. The higher intensity of antigen- antibody interaction for calreticulin and PDI A3 was observed in cancerous tissue compared to normal tissue, which may indicate the higher expression of these proteins in cancerous tissue.

Discussion

TNM staging is determined according to the outcomes of physical examination, biopsy, and imaging tests 7. It is useful to guide physicians in treatment option and prognosis of patients 9. In this study, we focused on the protein expression changes in N0, N1 and N2 stages of breast cancer. Due to limitation in sample size, N3 stage was not included in our study.

TLB buffer made up of thiourea, urea and other reagents that favor the extraction of membrane associated or membrane proteins was used to extract proteins from the tissue pellet that was subjected to prewashing with TRIS buffer for removal of hydrophilic proteins. Thirty percent of human proteome is consisted of membrane proteins 9, these proteins involve in various biological processes of cell, which include signal transduction, immune regulation and transportation 10. In addition, the nature of these proteins of being membrane associated making them good target for recognition in drug-targeted therapy for treatment of cancers.

In general, the differentially expressed proteins identified in this study can be grouped as either common proteins in all stages or predominant protein for each stage. The common proteins can be potentially used as biomarkers for diagnosis or treatment for all N stages. On the other hand, the predominant protein in each stage may be collectively used with other diagnoses for classification of N staging. Two common proteins that were up-regulated in all stages were calreticulin and tropomyosin alpha 3 chain. Calrecticulin was a very promising biomarker in view of its high expression consistency in most of the patients, where it was up-regulated significantly at 77%, 92% and 83% in N0, N1 and N2 stages, respectively.

Calreticulin was first introduced by Ostwald and MacLennan in 1974 11. It is a Ca2+ binding protein that localizes in the peripheral membrane of lumen of ER and nuclear envelope. It is important for Ca2+ storage, signaling and regulation in ER 12. As reviewed by Michalak et al., (1999), calreticulin is also a lectin-like chaperone that involves in the synthesis of a variety of molecules, such as ion channels, surface receptors, intergrins and transporters. It helps to increase the yield of correctly folded proteins and prevents the aggregation and assembly of partially folded proteins. It had been reported as potential biomarker for breast cancer correlated with postoperative metastasis 13, 14.

The protein that predominantly up-regulated (69%) in N0 stage was HSP 70, HSP 70 is a member of heat shock family that was induced under stress environment. It is function as ATP-dependent molecular chaperone 18, 19 that interacts with unfolded polypeptide sequences during mRNA translation to prevent premature self-association in nascent protein. It also assists in transportation of proteins across cellular membranes and targeting protein for lysosomal degradation 19. HSP 70 was reported up-regulated in breast cancer 20.

When comparing to other stages, N1 stage has the highest number of predominantly up-regulated proteins, namely 80 k protein H precursor, tropomyosin isoform, PDI, HSP 90 (or also known as tumor rejection antigen), tubulin, beta polypeptide and heat shock 70 kDa protein 1. Amongst these, 80 k protein H precursor was the most consistent up-regulated protein in N1 stage, where it was found up-regulated at 85%. This is followed by PDI at 76.9% while the rest of the proteins were up-regulated at 69%. Therefore, we foresee the usefulness of 80 k protein H precursor and PDI as promising markers that can be collectively used to indicate N1 stage of breast cancer. 80 k protein H precursor or also known as glucosidase 2 subunit beta, is a substrate for protein kinase C 21. It involves in the mechanism of elevated fibroblast growth factor-1 (FGF-1) in breast cancer 22, where FGF-1 is one of the key factors in angiogenesis and lymphangiogenesis in cancer 23. On the other hand, PD1 is an enzyme that catalyzes the formation, reduction and isomerization of protein disulfide bond in ER 24, 25, where it functions as molecular chaperone by assisting in protein folding. Hence, it plays a critical role in the maintenance of protein structural stability and functional integrity 26. In addition, PDI is also known as intracellular estrogen-binding protein, which accumulates estrogen in cells and augment the activities of estrogen receptor-mediated transcription 24. PDI was reported as a differentially expressed protein in IDC breast cancer 27.

The predominantly up-regulated protein for N2 stage was 78 kDa glucose-regulated protein, it was significantly up-regulated at 75%. Its alternate name is immunoglobulin heavy chain binding protein or “BIP”. Similar with heat shock family members, it functions as molecular chaperone that assists in folding and assembly of newly synthesized proteins. Its expression is induced under glucose starvation 28. It was reported up-regulated in breast cancer 29 and colorectal cancer 30.

It is interesting to note the significant up-regulation of PDI A3 was only found in N1 and N2 stages, which represent the breast cancer that had metastasized. In addition, its extent of up-regulation expression in N2 stage is worth mentioning, where it was up-regulated significantly at 91% in N2 stage while only at 69% in N1 stage. The up-regulation of PDI seems to be paralleled with the advancing of N stages (51% at N0 stage). We believe that PDI A3 may be used in the prognosis of breast cancer, where the presence of this protein may indicate the metastasize potential of breast cancer. PDI A3 is one of the members of PDI family. The thiol-disulfide exidoreductase activity of PDI A3 catalyzes oxidation, reduction and isomerization of protein disulfide bond. PDI A3 non-covalently interacts with two lectin molecular chaperones known as calnexin and calreticulin. PDI A3 involves in the oxidation folding of newly synthesized glycoproteins 25, 31, 32. It was also reported up-regulated in gastric cancer 33 and pancreatic cancer 34.

Conclusion

In this study, we detected two proteins, namely calreticulin and tropomysin alpha 3 chains as common up-regulated proteins in N0, N1 and N2 stages of breast cancer, while HSP 70, 80 k protein H precursor, PDI, and 78 kDa glucose-regulated protein were predominantly up-regulated proteins in N0, N1 and N2, respectively. On the other hand, the increase up-regulation pattern of PDI A3 was parallel with the advancing N stages of breast cancer. By virtue of cellular location of membrane proteins in cells, their up-regulation expression in cancerous tissues can potentially use as indicators for diagnosis or drug-targeted therapy of breast cancer, nevertheless, before all these potential biomarkers turn to be used in clinical applications, more retrospective and prospective clinical trials are needed to evaluate the overall clinical utility of these indicators. Furthermore, the significance of each biomarker for breast cancer can be evaluated by using a greater number of tissue specimens.

Acknowledgments

We thank Ministry of Science, Technology and Innovation Malaysia for providing a grant (Project number: 02-01-05-SF0353) to fund this project, the Ministry of Heath, Malaysia for providing the human ethical clearance for conducting this research. Special thanks to Malaysian Institute of Pharmaceuticals and Nutraceuticals for providing LC-MS/MS analysis instruments throughout the study.

28. Delpino A, Castelli M. The 78 kDa Glucose-Regulated Protein (GRP78/BIP) Is Expressed on the Cell Membrane, Is Released into Cell Culture Medium and Is Also Present in Human Peripheral Circulation. Bioscience Reports. 2002;22:407–420.[PubMed]